Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rabbit P450 2E1 was stably expressed in Chinese hamster ovary cells after cotransfection with pRC/CMV-2E1 and pFR400 which expresses murine dihydrofolate reductase with a single arginine to leucine substitution at position 22. This mutation permits amplification of expression with increasing methotrexate concentrations in CHO-K1 cells that are not dihydrofolate reductase deficient. After amplification with 1 microM methotrexate, a representative clone expressed about 15 pmol of P450 2E1/mg microsomal protein. Cells from a single 35-mm plate catalyzed the formation of 1.02 nmol 6-hydroxychlorzoxazone/10(6) cells/h or about 127 pmol/mg total cell protein/min. The enzyme was rapidly labeled when pulsed with [35S]-methionine. Initial pulse-chase experiments indicate that the expressed protein has a half-life of 4.8 h.
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PMID:Rabbit P450 2E1 expressed in CHO-K1 cells has a short half-life. 782 77

The untranslated first exon and the 5'-flanking region for the rat liver NADPH-cytochrome P450 oxidoreductase gene has been isolated from a Wistar-Furth genomic library. The remainder of the gene is composed of 15 exons which code for the mature protein and a 3'-nontranslated segment (T. D. Porter et al. Biochemistry, 1990, 29, 9814-9818). The 56-bp first exon resides 30.5 kb upstream from exon two, making the total gene length approximately 50 kb. While the region surrounding the start site (TCAGAGAC) was found to be homologous to a eukaryotic cap signal, the 5' flanking region possesses neither a TATA nor a CCAAT box. Instead it contains five GC-rich hexanucleotide consensus sequences for the transcription factor Sp1. These features clearly distinguish it from genes encoding other members of the mixed-function oxidase system, the cytochromes P450. Primer extension analysis and S1 nuclease mapping identified multiple transcriptional start sites. In many respects, the TATA-less oxidoreductase promoter resembles the promoter regions of dihydrofolate reductase and other housekeeping genes. Northern blot analysis demonstrates that this promoter is modulated by phenobarbital and trans-stilbene oxide, known inducers of oxidoreductase.
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PMID:NADPH cytochrome P-450 oxidoreductase gene: identification and characterization of the promoter region. 817 32

Some xenobiotics induce membrane-bound drug metabolizing enzymes (Xme) and a profound proliferation of the endoplasmic reticulum (ER) in vivo. However these effects are much weaker in vitro, possibly due to absence of certain transcription factors. We tested the possibility that ER proliferation can affect the level of ER-resident enzymes even in the absence of transcriptional activation. For this purpose we analysed the effects of compactin, which has been shown to induce ER proliferation in vitro, on recombinant Xme, which were expressed from a constitutive viral promoter. High levels of recombinant UDP-glucuronosyltransferase UGT1A6 were achieved by amplification of the UGT1A6 cDNA using the dihydrofolate reductase cDNA as selectable marker in DHFR- CHO cells. Treatment of the resulting cell lines with lipoprotein-deficient serum in the absence and presence of compactin for 5 days resulted in a 1.3- and 2.3-fold, respectively, increase of the UGT enzyme activity towards 4-methylumbelliferone, paralleled by an induction of immunoreactive UGT1A6 protein. Similarly, treatment with this 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor increased the endogenous P450 reductase activity 2.6-fold, concomitant with an increase of immunodetectable protein. As expected compactin induced the level of 3-hydroxy-3-methylglutaryl-CoA reductase. Increased levels of this protein have been associated with a proliferation of the ER. Compactin treatment of a separate cell line that expressed recombinant human P450 reductase increased this enzyme activity fivefold. Pulse-chase experiments revealed that the induction of the recombinant Xme by compactin was most likely due to decreased protein degradation. Our results show that enzyme systems unrelated to those involved in cholesterol biosynthesis are affected by compounds known to affect membrane biogenesis. Since this effect extends to heterologously expressed enzymes, it also provides an efficient means by which to increase the levels of recombinant ER proteins.
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PMID:Treatment of mammalian cells with the endoplasmic reticulum-proliferator compactin strongly induces recombinant and endogenous xenobiotic metabolizing enzymes and 3-hydroxy-3-methylglutaryl-CoA reductase in vitro. 991 63

NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways.
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PMID:Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways. 1136 92